Apoptotic cell death induced by chemotherapeutics and other cell-damaging agents typically involves release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm, where it induces the oligomerization of Apaf-1 to form the apoptosome and activate procaspase 9. Active caspase 9 then activates the executioner caspases 3 and 7 to dismantle the doomed cell. Impaired function or formation of the apoptosome is a hallmark of many cancers, including ovarian cancers, melanoma, lung cancers and others. We have demonstrated that apoptosome formation is defective in leukemias expressing activated tyrosine kinases (Bcr-Abl, Tel-PDGFR2, and activated FLT3). This defect was traced to tight inhibitory binding of the beta isoform of Hsp90 to Apaf-1 in leukemic, but not normal cells. Specifically, a difference in the phosphorylation status of Hsp902 at two sites (S226/S255) could account for the difference in Apaf-1 binding and cytochrome c-induced caspase activation between leukemic and untransformed cells. Moreover, expression of a non- phosphorylatable Hsp902 variant that mimicked the leukemic form conferred resistance to cytochrome c-induced apoptosis in untransformed cells and promoted resistance to the front-line therapeutic imatinib in Bcr-Abl-transformed bone marrow-derived hematopoietic stem cells in vitro. These findings point to the kinases/phosphatases regulating Hsp90 as potentially attractive therapeutic targets. The aims of this proposal are to elucidate how Hsp902 phosphorylation is misregulated in leukemic cells, to determine how Hsp902 hypophosphorylation impacts Apaf-1 and other Hsp902 targets, to analyze the effects of Hsp902 hypophosphorylation in animals and to evaluate Hsp902 phosphorylation status in patient samples. The long term goal of this work is to fully understand the molecular basis for apoptosome resistance in leukemia and, ultimately, to translate this knowledge into therapeutic agents for leukemias.